The use of the same fixed therapeutic range for all aPT reagents is problematic because the responsiveness of different aPT reagents to heparin is highly variable [4-8]. Therefore, a therapeutic range of 1.5 to 2.5 times the control value reflects different dosages of heparin and different heparin levels when different aPT reagents are used. The problem of standardizing aPT reagents is similar to that for standardizing prothrombin time reagents but has not been solved by using the equivalent of the International Normalized Ratio [9]. Although the replacement of unfractionated heparin by low-molecular-weight heparin may circumvent the problem of laboratory monitoring, experience with low-molecular-weight heparin in the treatment of thrombosis is limited [10, 11]. In addition, these new anticoagulants are not approved for treatment of venous thrombosis in North America. For the foreseeable future, unfractionated heparin will be used for the treatment of thrombosis, and aPT standardization will remain a major problem requiring resolution.

To determine the extent of the variability of the therapeutic ranges for different aPT reagents and the potential clinical importance of this variability, we did comparative studies on plasma samples from patients who were being treated with heparin. Because a heparin level of 0.2 to 0.4 U/mL by protamine titration assay has been shown to correlate with clinical efficacy and safety both in clinical [12-14] and experimental animal [15] studies, we defined the therapeutic range for aPT results of the different reagents as that which corresponded to a heparin level of 0.2 to 0.4 U/mL. In this report, we discuss our results and present an approach for standardizing the reporting of aPT results in patients treated with heparin.

Methods

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Venous blood was collected in Vacutainer tubes (Becton Dickinson, Mississauga, Ontario, Canada) containing 3.2% buffered sodium citrate. To obtain platelet-free plasma, the citrated sample was centrifuged for 15 minutes at 1500g. The plasma was then placed in a plastic tube, centrifuged again for 5 minutes, and pipetted into a clean polypropylene tube. It was frozen at –70°C for later measurement of heparin levels and aPT assays. Heparin levels were determined by protamine titration according to the method of Refn and Vestergaard [16].

Four conventional aPT reagents were used: 1) Dade Actin FS [Baxter Diagnostics, Inc., Mississauga, Ontario, Canada]; 2) Activated Thrombofax [Ortho Diagnostic Systems, Markham, Ontario, Canada]; 3) Auto aPT [Organon Teknica, Inc., Scarborough, Ontario, Canada]; and 4) Instrumentation Laboratories aPT (Fisher Scientific, Unionville, Ontario, Canada). The aPT tests were done according to the manufacturer's instructions, using the Automated Coagulation Laboratories 300R or 810 instrument (Fisher Scientific). In addition, in one study (study 2), the Ciba Corning 512 Monitor (Ciba Corning Diagnostics Corporation, Medfield, Massachusetts) was used to determine the plasma-equivalent aPT from whole venous blood as described by Ansell and colleagues [17].

The coagulation assays were done on plasma samples from patients with venous thrombosis who were being treated with heparin. Study 1 was done to evaluate the reproducibility of different batches of the same brand of reagent obtained from a single manufacturer during a period of 7 years. This evaluation was achieved by assessing the relation between the aPT results and heparin levels on plasma samples at three different times (1984, 1988, and 1991) using four different lots of Dade Actin FS reagent.

Study 2 was done to evaluate the relation between the aPT results and heparin levels using aPT reagents from several manufacturers. This study was done in 1991 on plasma samples from 70 patients with venous thrombosis using Activated Thrombofax (Ortho), Instrumentation Laboratories aPT (IL), Auto aPT (Organon), and Ciba Corning plasma-equivalent aPT (Biotrack). The aPT results were compared with simultaneously measured heparin levels.

Patients

For each reagent, the control values were determined by calculating the mean aPT results for plasma samples obtained from a minimum of 20 hospital personnel who had no coagulation defects and were receiving no medications. Patients involved in studies 1 and 2 were inpatients who had either venous thrombosis or pulmonary embolism and who were receiving continuous infusions of unfractionated heparin intravenously for at least 6 hours after a bolus dose of 5000 U. None of the patients was receiving warfarin at the time of blood sampling.

Plasma samples for study 1 patients were obtained in 1984 (52 patients; mean heparin level, 0.21 U/mL); in 1988 (73 patients; mean heparin level, 0.30 U/mL); and in 1991 (66 patients; mean heparin level, 0.24 U/mL). Plasma samples for study 2 were obtained from the same pool of 70 patients treated with heparin in 1991. The mean heparin level was 0.26 U/mL. The aPT reagents tested were Ortho (57 patients), IL (52 patients), Biotrack (65 patients), and Organon (55 patients).

Analysis and Statistics

For each reagent, the relation between the aPT results and ex-vivo heparin levels (both derived from aliquots of the same plasma sample) was analyzed by linear regression. Comparisons among regression lines of various reagents (either different lots or different manufacturers) were achieved through multiple linear regression, first by comparing slopes and then by comparing intercepts in a model constrained to have a common slope [18]. The target range of aPT results that corresponds to protamine titration heparin levels of 0.2 to 0.4 U/mL Figure 1 was derived as the ordinate (Y axis) value for the point on the regression line at these two heparin levels (X axis). A P value of less than 0.05 was considered to be statistically significant.

View larger version (17K): [in this window] [in a new window]   Figure 1. Therapeutic ranges for Dade Actin FS (top panel) and Ortho Activated Thrombofax (bottom panel) activated partial thromboplastin time (aPT) reagents. The therapeutic range of aPT values is determined by the points (dotted lines) on the regression line (solid line) that correspond to the protamine titration heparin level of 0.2 to 0.4 U/mL. See Methods for manufacturing information.

Results

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Study 1

The regression lines for aPT results and corresponding heparin levels for the Dade reagent—lots 13 (1984), 102 (1988), 129 (1991), and 154 (1991)—are shown in Figure 2. A formal comparison of the slopes of the four regression lines indicated little evidence of nonparallelism (P > 0.2) but strong evidence of differences in intercept (P < 0.001). The visual impression that lot 154 is different is supported by additional comparisons of intercepts that show that lots 13, 102, and 129 are similar (P > 0.2), whereas comparisons of lot 154 with the other three intercepts show that lot 154 differs (P < 0.001). In addition, the intercept of lot 154 differed from lot 129 despite use of duplicate plasma samples from the same group of patients studied in 1991 (P < 0.001). We considered the possibility that lot 154 was simply an outlier, and we received two replacement reagents from the manufacturer (lots 147 and 149) that were produced in the same period as lot 154. A comparison of lot 154 with lots 147 and 149 yielded regression lines that were not significantly different (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 2. A comparison of regression lines describing the relation between the activated partial thromboplastin time (aPT) and heparin levels for Dade Actin FS reagents over several years. The slopes of the regression lines are similar (P > 0.2). The intercepts for lots 13 (1984), 102 (1988), and 129 (1991) are similar (P > 0.2), but the intercept for lot 154 (1991) differs from the other lots (P < 0.001). See Methods for manufacturing information.

Study 2

The regression lines for the Ortho, Biotrack, IL, and Organon reagents are shown in Figure 3. The visual suggestion of differences in the slope of these regression lines was confirmed by the formal statistical analysis. The null hypothesis of all relations in Figure 3 being parallel was rejected (P = 0.009), indicating evidence of real differences in the slopes of the lines. An additional test of the null hypothesis that all the regression lines were coincident was even more untenable (P < 0.001), indicating that the intercepts, in addition to slopes, also vary among the reagents from different manufacturers.

View larger version (16K): [in this window] [in a new window]   Figure 3. Comparison of the regression lines describing the relation between the activated partial thromboplastin time (aPT) and heparin levels for various manufacturers' reagents. The slopes of the regression lines differ (P = 0.009), as do the intercepts (P < 0.001), among the various manufacturers' reagents. See Methods for manufacturing information.

The Effect of Different aPT Reagents on the Therapeutic Range

The results for study 1 are shown in Table 1. The control values, determined from a group of at least 20 normal patients, remained similar over time for each lot of the Dade reagent. The target therapeutic ranges, therefore, which correspond to an aPT ratio of 1.5 to 2.5, are similar for the different lots. In contrast, however, the ex-vivo heparin level corresponding to an aPT ratio of 1.5 (the lower limit of the therapeutic range) varies considerably among reagents. For all lots of the Dade aPT reagent studied, the heparin level corresponding to an aPT ratio of 1.5 is much less than 0.2 U/mL (<0.05, 0.05, 0.07, and 0.10 U/mL).

The results of study 2 are shown in Table 2. As in study 1, the therapeutic ranges based on aPT ratios are similar for each reagent. However, an aPT ratio of 1.5 times the control value for each of the four reagents corresponds to heparin levels that are considerably less than the targeted lower limit of 0.2 U/mL (Ortho and Biotrack, 0.09 U/mL; Organon and IL, 0.08 U/mL). For the Biotrack, IL, and Organon reagents, an aPT ratio of 2.5 times the control value (the upper limit of the therapeutic range) corresponds to a heparin level greater than 0.2 U/mL, but for the Ortho reagent, an aPT ratio of 2.5 corresponds to a heparin level less than 0.2 U/mL.

Discussion

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Historically, the concept of using a therapeutic range for heparin was derived from experimental animal studies in which the whole-blood clotting time was used [19, 20]. This clotting time was replaced by the aPT as a measure of the intrinsic pathway of coagulation and was subsequently used to monitor heparin therapy [21]. In 1972, Basu and colleagues [22] did a prospective study of patients with venous thromboembolism who were treated with heparin. They reported that the risk for recurrent thromboembolic disease was associated with failure to obtain an aPT ratio of approximately 1.5 times the control value. This aPT ratio then became the standard for the lower limit of the therapeutic range.

The use of a fixed therapeutic range for the aPT is now known to be problematic. For example, if a heparin level of 0.2 to 0.4 U/mL is used as a reference standard for the therapeutic range, then the corresponding aPT range varies from 65 to 100 seconds for the Biotrack reagent to 100 to 160 seconds for the Ortho reagent. On the other hand, if the therapeutic range is based on an aPT ratio of 1.5 to 2.5 for each reagent, then the heparin levels vary from 0.09 to 0.24 U/mL (Biotrack) to 0.09 to 0.018 U/mL (Ortho).

It is unlikely that the results observed are caused by the laboratory instrument; a previous study [23] showed that the responsiveness of the reagent, not of the instrument, accounts for the variation in response to heparin. No additional variable was used in the comparisons of lots. The most important confounder that could alter the relation between the aPT and heparin levels is the coagulation factor levels (in particular, Factor VIII and fibrinogen). These levels were not measured and therefore were not used in the analysis. There are two reasons that factor levels probably did not account for the observations in this study. First, comparisons of the same reagent (study 1) were done over several years, using different groups of plasma samples. This technique favors variation caused by differing levels of coagulation factors; however, as we described, the relation between the aPT and heparin levels was similar for three of the four lots tested, indicating that different coagulation factor levels were not the cause of the variation. Second, the comparisons of several manufacturers' reagents (study 2), done on identical plasma samples with the same factor levels, showed variation, again providing evidence that factor levels did not influence the relation between the aPT and heparin levels.

The practical implications of these findings are that a different heparin dose would be required to produce an aPT ratio of 1.5 with different reagents and that, for all of the reagents tested, an aPT ratio of 1.5 is subtherapeutic. Although suggesting a ratio of aPT results of 2 to 3 times the control value may encompass therapeutic heparin levels for some reagents, no assurance can be given that, with changes in the manufacturing process and introduction of new reagents, such a recommendation is reasonable. Furthermore, our study of a single manufacturer's reagent (Dade) shows that it is difficult to produce aPT reagents that have identical responsiveness to heparin.

No aPT standard exists, and, as shown in our study and in others [4-8], the responsiveness of different commercial aPT reagents to heparin is variable. We chose the heparin level assayed by protamine titration as our reference standard. The use of a heparin level of 0.2 U/mL as the lower limit of the therapeutic range is justified by the results of experimental animal studies and of clinical trials. In 1977, Chiu and colleagues [15], using a venous thrombosis model in rabbits, showed that accretion of fibrin onto an existing thrombus was inhibited when heparin was administered to achieve a plasma heparin level of 0.2 U/mL (measured by protamine titration). In this study, a heparin level of 0.2 U/mL corresponded to an aPT of 1.5 times the control aPT value. In addition, no further inhibition of fibrin accretion took place at levels greater than 0.4 U/mL, but substantial bleeding was observed with heparin levels greater than 0.5 U/mL.

The principle that a heparin level of 0.2 U/mL corresponds to a minimum effective anticoagulant dose is supported by the results of four clinical trials—three in patients with venous thrombosis and one in patients with acute myocardial infarction. In the first study [12]—a randomized trial that compared heparin administered either by continuous infusion or subcutaneous injections in patients with proximal deep venous thrombosis—subgroup analysis showed that there was a 15-fold increase in recurrence of venous thrombosis if the early anticoagulant effect was subtherapeutic. A response was defined a priori as subtherapeutic if the aPT ratio was less than 1.6, which corresponded to an ex-vivo heparin level of 0.2 U/mL (by protamine titration).

In the second study [13], which compared a long course with a short course of heparin therapy, the targeted therapeutic range for the aPT was based on an ex-vivo heparin assay of 0.2 to 0.4 U/mL by protamine titration. This therapeutic range for the aPT was established before doing the study, using the methods we have outlined here. The dose of heparin was adjusted according to this range [24], and the incidence of major bleeding and recurrent thromboembolic disease was low (approximately 7% in both groups).

In the third study (Levine and colleagues. Unpublished data.), patients requiring higher-than-average doses of heparin (>35 000 U per 24 hours) were randomly assigned to be monitored by either an aPT or anti-Factor Xa heparin level, both equivalent to a heparin level of 0.2 U/mL by protamine titration. The rates of recurrent thrombosis (4%) and bleeding (2%) were low in both groups. Finally, in a subgroup analysis of a study comparing fixed regimens of high- and low-dose heparin in patients with recent anterior-wall myocardial infarction [14], a heparin level of approximately 0.2 U/mL (by protamine titration) distinguished patients with from those without mural thrombi in the high-dose heparin group.

Standardization of the aPT is critical for optimal patient care but has been difficult to achieve [25]. Our approach for the last 8 years has been to establish a therapeutic range for each new lot of aPT reagent as follows: 1) Collect and freeze plasma samples prepared from blood drawn no less than 4 hours after a bolus dose from at least 30 patients with thrombosis who are chosen at random and who are receiving heparin therapy [and not receiving concomitant warfarin]; 2) measure protamine titration heparin levels and do aPT assays on each sample using the new lot of partial thromboplastin reagent; 3) plot the relation between the heparin levels and the aPT results using the least-squares regression technique and calculate a line of best fit from the regression equation; and 4) use as the therapeutic range the ordinate (Y axis) value of the point on the regression line that corresponds to heparin levels of 0.2 to 0.4 U/mL by protamine titration. The calculated aPT therapeutic range can be inserted into a nomogram for heparin dosing [24, 26]. During the period of 8 years, using the same manufacturer's reagent, our range changed from 62 to 80 seconds to 79 to 105 seconds.

Although it would be more convenient to compare aPT assays with heparin levels by using samples of plasma to which a range of heparin concentrations have been added in vitro, such an approach may not be appropriate. We use the standardization procedure on plasma obtained from patients treated with heparin because the relation between the aPT and heparin levels is different when measured on plasma from patients treated with heparin than it is when the assays are done on plasma to which heparin has been added [4, 6, 7, 25]. This difference in the relation between the aPT and heparin levels probably occurs because the clearance of the high-molecular-weight fractions is more rapid than that of the lower molecular-weight moieties [27, 28]. As a result, the profile of the molecular-weight distribution of heparin, and therefore the relation between the aPT and heparin levels, is different ex vivo than in vitro. Furthermore, the evidence supporting the use of a therapeutic range is based on studies using ex-vivo heparin level assays as a reference standard [12-14].

Although many hospitals can determine the therapeutic range for each new lot of aPT reagent as we have described, the procedure might be difficult to do in small institutions with no ready access to plasma samples from patients treated with heparin. The transition from an old to a new reagent would be simplified if manufacturers standardized each new batch of thromboplastin reagent and provided laboratories with a therapeutic range for the aPT equivalent to a heparin level of 0.2 to 0.4 U/mL, based on protamine titration.